Christopher T. Schafer

Chemokines and their receptors mediate cell migration and coordinate immune responses, while dysregulation can lead to inflammation. Therapeutic modulation of the chemokine signaling axis has proven difficult. Most drug discovery efforts target the receptors, whereas natural regulatory mechanisms modulate immune responses by directly binding to or modifying the chemokine ligands. Despite this insight, development of effective chemokine-directed modulators has remained elusive. Recent advances in de novo protein design offer an unprecedented opportunity to produce high-affinity binders that efficiently block protein-protein interactions. We implemented a computational workflow leveraging the BindCraft platform to generate miniprotein binders against CCL25, the chemokine ligand for the receptors CCR9 and ACKR4 and implicated in inflammatory bowel diseases. The unbiased development results in several miniproteins designed to block the receptor N-terminus from wrapping the chemokine and prevent productive engagement. Thus, these proteins suppress CCL25-mediated effector coupling and halt MOLT-4 lymphoblast migration. Another class of miniprotein, represented by VUP25111, is predicted to bind CCL25 along the chemokine β1 strand and retained receptor binding. This complex inhibited arrestin recruitment to CCR9, but not to ACKR4, indicating receptor specificity. Additionally, G protein recruitment to CCR9 was unimpeded by VUP25111, suggesting that the miniprotein biased the native agonist towards G proteins. These results demonstrate the effectiveness of differentially targeting CCL25 to suppress CCR9 signaling and new tools to resolve the structural basis of chemokine receptor activation and bias.

Chemokine receptors are G protein–coupled receptors (GPCRs) that drive cell migration through interactions with chemokines. Even in the absence of ligand, chemokine receptors spend a portion of their time in active conformational states, rendering them constitutively active. Here, we identified and characterized two mutations in the third intracellular loop of atypical chemokine receptor 3 (ACKR3) that inhibited or promoted its constitutive activity as assessed by ligand-independent β-arrestin recruitment and receptor internalization. Single-molecule fluorescence resonance energy transfer experiments showed that these effects on activity corresponded to changes in the conformational equilibrium of the receptor, which also affected the kinetics of chemokine binding. The potencies of chemokines and small-molecule agonists for β-arrestin recruitment were differentially affected by the mutations, showing that the effects of an altered receptor conformational equilibrium on receptor activation were ligand dependent. Together, our findings reveal an inside-out allosteric mechanism for ACKR3 in which intracellular, conformation-altering mutants affect the ability of extracellular ligands to bind to and activate the receptor.

The atypical receptor ACKR3 works together with the canonical chemokine receptor CXCR4 to drive cell migration along gradients of their shared agonist CXCL12. CXCR4 promotes chemotaxis by activating canonical G protein pathways and recruiting β-arrestins. ACKR3 indirectly regulates CXCR4-mediated chemotaxis by scavenging CXCL12. Unlike canonical chemokine receptors, ACKR3 does not couple to G proteins and instead is 100% biased towards β-arrestins. CXCR4 activation by CXCL12 is exquisitely sensitive to subtle changes in both receptor and ligand. By contrast, ACKR3 is activation-prone: it recruits β-arrestins in response to many ligands and is much less sensitive to mutations, suggesting distinct activation mechanisms compared to CXCR4. To explore the basis of these differences, we compared the dynamics of ACKR3 and CXCR4 complexes with chemokines using molecular dynamic (MD) simulations. Ten-microsecond atomistic MD simulations revealed that CXCR4 adopts a stable active state when bound to WT CXCL12 but transitions to an inactive state when in complex with the antagonist variant, [P2G]CXCL12. By comparison, ACKR3 exhibits a variable transmembrane (TM) 6 state distribution and persistently “active” TM7 when complexed with either WT CXCL12 or [P2G]CXCL12, the latter retaining substantial agonistic activity at ACKR3. We further identified ligand-mediated residue interaction networks in the TM core that regulate TM6 and TM7 activation in CXCR4 but are absent or disrupted in ACKR3, resulting in less constrained receptor dynamics. These findings were validated by BRET-based assays with CXCL12 and ACKR3 mutants. Together, the data suggests that the unique conformational dynamics of ACKR3 govern its activation propensity, its ligand promiscuity, and its atypical effector coupling.

Chemokines and their receptors mediate cell migration and coordinate immune responses, while dysregulation can lead to inflammation. Therapeutic modulation of the chemokine signaling axis has proven difficult. Most drug discovery efforts target the receptors, whereas natural regulatory mechanisms focus on the chemokines. Despite this insight, development of effective chemokine-directed modulators has remained elusive. Recent advances in de novo protein design offer an unprecedented opportunity to produce high-affinity binders that efficiently block protein-protein interactions. We implemented a computational workflow leveraging the BindCraft platform to generate miniprotein binders against CCL25, the chemokine ligand for the receptors CCR9 and ACKR4 and implicated in inflammatory bowel diseases. The unbiased development results in several miniproteins designed to block the receptor N-terminus from wrapping the chemokine and prevent productive engagement. Thus, these proteins suppress CCL25-mediated effector coupling and halt MOLT-4 lymphoblast migration. Another class of miniprotein, represented by VUP25111, is predicted to bind CCL25 along the chemokine β1 strand and retained receptor binding. This complex inhibited arrestin recruitment to CCR9, but not to ACKR4, indicating receptor specificity. Additionally, G protein signaling through CCR9 was unimpeded by VUP25111, suggesting that the miniprotein biased the native balanced agonist towards G proteins. These results demonstrate the effectiveness of differentially targeting CCL25 to suppress CCR9 signaling and new tools to resolve the structural basis of chemokine receptor activation and bias.

Chemokines and their receptors mediate cell migration and coordinate immune responses, while dysregulation can lead to inflammation. Therapeutic modulation of the chemokine signaling axis has proven difficult. Most drug discovery efforts target the receptors, whereas natural regulatory mechanisms focus on the chemokines. Despite this insight, development of effective chemokine-directed modulators has remained elusive. Recent advances in de novo protein design offer an unprecedented opportunity to produce high-affinity binders that efficiently block protein-protein interactions. We implemented a computational workflow leveraging the BindCraft platform to generate miniprotein binders against CCL25, the chemokine ligand for the receptors CCR9 and ACKR4 and implicated in inflammatory bowel diseases. The unbiased development results in several miniproteins designed to block the receptor N-terminus from wrapping the chemokine and prevent productive engagement. Thus, these proteins suppress CCL25-mediated effector coupling and halt MOLT-4 lymphoblast migration. Another class of miniprotein, represented by VUP25111, is predicted to bind CCL25 along the chemokine β1 strand and retained receptor binding. This complex inhibited arrestin recruitment to CCR9, but not to ACKR4, indicating receptor specificity. Additionally, G protein signaling through CCR9 was unimpeded by VUP25111, suggesting that the miniprotein biased the native balanced agonist towards G proteins. These results demonstrate the effectiveness of differentially targeting CCL25 to suppress CCR9 signaling and new tools to resolve the structural basis of chemokine receptor activation and bias.

Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein-coupled receptor (GPCR) kinases (GRKs) to efficiently control chemokine levels. Despite not coupling G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. Here we resolve the underlying mechanisms allowing ACKR4 to circumvent the G protein requirement for GRK3 function. Using live cell BRET assays, we confirm that ACKR4 is preferentially phosphorylated by the GRK2/3 kinase family and that both GRK recruitment and receptor phosphorylation occur in the absence of activated G proteins. Instead, the kinases are recruited directly by a unique acidic rich motif in the proximal receptor C-terminus which coordinates productive phosphorylation reactions. Mutations in this region severely attenuated kinase recruitment and phosphorylation. Productive phosphorylation reaction plays a substantial role in the G protein-independent mechanism and a ‘kinase-dead’ GRK3 (KD-GRK3) has severely reduced recruitment to ACKR4. This was not observed for KD-GRK3 translocation to GPCRs that recruit the kinase in a G protein-dependent manner. Together, these findings suggest that ACKR4 directly coordinates GRK3 recruitment and phosphorylation, highlighting a uniquely evolved atypical mechanism to utilize GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor.

Stimulation of atypical chemokine receptor 3 (ACKR3) by chemokines does not activate G proteins but recruits arrestin. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive responses of ACKR3 using inverse agonistic nanobodies to suppress its basal activity. These tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal non-chemotactic, cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide an alternate therapeutic approach for targeting ACKR3.

Unique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote distinct cellular outcomes¹, but it is unclear whether or how arrestins differentially engage these barcodes. Here, to address this, we developed an antigen-binding fragment (Fab7) that recognizes both active arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) without interacting with bound receptor polypeptides. We used Fab7 to determine the structures of both arrestins in complex with atypical chemokine receptor 3 (ACKR3) phosphorylated in different regions of its C-terminal tail by either GRK2 or GRK5 (ref. ²). The GRK2-phosphorylated ACKR3 resulted in more heterogeneous 'tail-mode' assemblies, whereas phosphorylation by GRK5 resulted in more rigid 'ACKR3-adjacent' assemblies. Unexpectedly, the finger loops of both arrestins engaged the micelle surface rather than the receptor intracellular pocket, with arrestin3 being more dynamic, partly because of its lack of a membrane-anchoring motif. Thus, both the region of the barcode and the arrestin isoform involved can alter the structure and dynamics of GPCR-arrestin complexes, providing a possible mechanistic basis for unique downstream cellular effects, such as the efficiency of chemokine scavenging and the robustness of arrestin binding in ACKR3.

The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G-protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.